The hypothesis that p53 deficiency enhances survival of DNA-damage bearing cells was investigated in embryonic stem (ES) cells with inactivation of one or both endogenous <I>p53</I> genes. ES cells were treated with a variety of DNA-damaging agents including UV light, γ-radiation and the topoisomerase I and II inhibitors, camptothecin and etoposide. Both topoisomerase inhibitors rapidly induced high levels of apoptosis in wild-type ES cells resulting in loss of most of the cell population within 48 hours. Consistent with results previously obtained from other cell types, the apoptotic response to etoposide was found to be p53-dependent at low doses but at the highest dose used p53-independent pathways became evident. Following UV-C irradiation, p53-protein was rapidly induced in wild-type cells and p53-dependent apoptosis followed within 8 hours, leading to death of the majority of cells within 36 hours. Treatment with ionising radiation led to enhanced expression of p53 but resulted in little induction of apoptosis irrespective of p52 status. Evasion of apoptosis in the short term does not necessarily imply a continued capacity for growth and therefore long term survival, measured by clonogenic potential, was also examined. Following both UV and γ-irradiation, clonogenic survival of <I>p53</I>-null cells was significantly higher than survival of wild-type cells, particularly after high levels of DNA-damage where survival was enhanced more than ten-fold. The data confirm that p53 restricts the numbers of cells bearing mutations that survive DNA damage induced by either agent, albeit by mechanisms that differ.

The transcription factor Nrf2 is a master regulator of cytoprotective gene expression. Nrf2 is negatively controlled by Keap1, a sensor protein which allows Nrf2 to respond to changing cellular conditions. In the basal state, Nrf2 binds to two sites of a Keap1 dimer allowing its ubiquitination in a Cullin-3/Rbx1-dependent manner. In response to electrophiles and oxidants (termed inducers, which bind directly to Keap1) ubiquitination of Nrf2 is inhibited; consequently, Nrf2 accumulates and activates transcription.We have developed aFLIM-based assay to study the dynamic interaction between Keap1 and Nrf2 in single live cells. Combinations of wild type and mutant proteins revealed that under basal conditions the Keap1-Nrf2 complex exists in two conformations, one in which Nrf2 is bound to both members or the Keap1 dimer (‘closed’ conformation), and a second in which Nrf2 interacts with a single Keap1 monomer (‘open’ conformation). We found that following exposure to a range of inducers the Cul3-Keap1-Nrf2 complex does not dissociate, but remains intact. Furthermore, we found that inducers lead to the accumulation of the Keap1-Nrf2 complex in the ‘closed’ conformation. Interestingly, blockage of the proteasome also leads to the accumulation of the complex in the closed conformation, suggesting that the binding of Nrf2 and its subsequent Keap1-dependent ubiquitination follows a cyclical pattern. We believe that the existence of a Keap1-Nrf2 binding cycle benefits the cell, as it allows other signaling pathways, such as those mediated by p21 and p62, to regulate Nrf2 activity in the absence of inducers. Together our results show that the interaction between Keap1 and Nrf2 is more dynamic than previously anticipated and that inducers function to modulate this dynamism, leading to Nrf2 stabilisation and cytoprotective gene expression.

Cortical and trabecular bone characteristics can be used to make predictions regarding previous loading regimes and developmental milestones which a bone has encountered. This has led to the suggestion that in the adult pelvis, bone patterning is related to the remodeling forces generated during bipedal locomotion. However, during the neonatal period the pelvic complex is non-load bearing, therefore, structural organisation of the ilium cannot reflect direct stance related forces. This study considers the cortical and trabecular bone structure in the ilium of the fetal and newborn infant, a structural configuration which until now has remained largely neglected in the literature. Only recently, with the advent of imaging modalities, has a greater insight and understanding of previously unexplored human bone structural composition and developing bone structure been made possible. In this study, multiple imaging techniques were applied to establish the optimal modality for application to the assessment of bone microstructure. Plain plate macroradiography and micro-computed tomography were identified as the gold standard imaging modalities for bone structural analysis for respective qualitative and quantitative assessment. These techniques were applied to gain a perspective of bone form from a sample of fetal and neonatal ilia selected from the Scheuer collection of juvenile remains. Initially, qualitative analysis highlighted consistent and well-defined patterns of cortical and trabecular bone organisation within the fetal and neonatal ilium, which corresponded with previously recognised regions in the adult that have been attributed directly to forces associated with bipedal locomotion. This was highly unexpected as the early developmental ilium is non-load bearing. Subsequently, quantification of the neonatal cortical and trabecular structure reinforced radiographic observations by identifying regions of significant architectural arrangement. Further investigation of this precocious patterning led to a revised proposal for the mode of growth in the human ilium during the neonatal developmental period. Analysis revealed statistically significant differences in regional trabecular characteristics and cortical thicknesses which have formed the basis of a proposed growth model for the ilium. The presence of ‘progressive growth regions’ and ‘restricted growth regions’ which appear to relate to metaphyseal and non-metaphyseal borders of the ilium have been demonstrated. Analysis of the early iliac bone pattern is important for understanding the relationship between trabecular bone patterning and cortical bone structure during the earliest stages of development in response to the specific functional forces acting during this period. It is suggested that the seemingly organised rudimentary scaffold observed in the early developmental ilium may be attributable to early ossification patterning, non-weight bearing anatomical interactions or even to a predetermined genetic blueprint. It must also be postulated that whilst the observed patterning may be indicative of a predetermined inherent template, early non-load bearing locomotive influences may subsequently be superimposed upon this scaffolding and perhaps reinforced and likely remodelled at a later age. Ultimately, the analysis of this fundamental primary pattern has core implications for understanding the earliest changes in iliac trabecular architecture and provides a baseline insight into future ontogenetic development and bipedal capabilities. Finally, the structural data and statistical analysis presented challenge the current concept of implied centrifugal ossification within the human ilium and present evidence of an alternative pattern of ossification that is largely dictated and controlled by basic anatomical principles.

No description available.

No description available.

The regulatory pathways involved in the rapid response of the AP-I transcription factor component, c-fos, to mechanical load in human primary osteoblast-like (HOB) cells and the human MO-63 bone cell line were investigated using a four-point bending model. HOB and MO-63 cells showed up regulation of c-fos expression after I-hour loading on fibronectin and collagen type I substrates; however, MO-63 cells did not respond on laminin YIGSR substrates. It was suggested that RGD mediated integrin interactions might be non-essential for the mechanical induction of c-fos at certain time points as indicated by a lack of inhibition associated with ROD-peptide treatment (however, evidence of the inhibitory nature of soluble RGD-peptides at these time points may require further examination). β1 integrin mediated interactions are critical as induction was completely blocked by anti-β1 integrin antibodies. The role of calcium signalling pathways was demonstrated by blocking up regulation with addition of EGTA, which chelates extracellular Ca2+, and gadolinium, which inhibits stretch-activated channels. L-type calcium channels may also be contributory but not essential, as addition of the L-type channel blocker, nifedipine inhibited the response, but not completely. Further evidence for involvement of calcium pathways followed addition of the Ca2+ ionophore A-23187 that induced temporally identical up regulation without loading. Protein kinase (C) mediated pathways were also shown to be critical, as shown by abolition of the response following H7 -dichloride treatment. Prostaglandin signalling pathways were non-essential, but were implicated in maximising load induction of c-fos as indicated by indomethacin induced inhibition. C-fos promoter analysis indicated that the major CRE is not essential for mechanically induced transcriptional activation of c-fos. An SRE containing promoter fragment appears to play a key role in this induction but is also not essential, indicating that multiple response elements are required. These results therefore demonstrate the essential nature of calcium, integrin and protein kinase mediated pathways in the induction of the early transcriptional mechanoresponse of bone, which are mediated by multiple response elements in the c-fos promoter.

A tissue-engineered small diameter blood vessel was developed by seeding endothelial cells onto a collagen matrix that was cross-linked and contracted by smooth muscle cells onto a polyester scaffold. Endothelial cells (EC) and smooth muscle cells (SMC) were isolated using enzyme dissociation of porcine aorta. Batches of these cells were accredited by immunohistochemical characterisation and functional assay prior to being used in any experiments. Once deemed suitable, they were cryopreserved for later use. Initial experiments determined the optimum SMC and collagen concentrations for producing a cellular rich collagen matrix. The use of a synthetic permanent polyester scaffold provided structural integrity for the developing blood vessel. Dedicated and robust bioreactor vessels and a pulsatile flow circuit were developed to be able to sustain the biologically active tissue and maintain physiological homeostasis. The retention of radiolabelled endothelial cells seeded onto the luminal surface of the grafts was assessed under conditions of high shear stress. The effect of surface modification using a variety of extracellular matrix proteins and low shear stress preconditioning of the SMC matrix was also assessed for its ability to enhance EC retention. Invivo evaluation of these tissue engineered grafts using a porcine model of infrarenal aortic implantation was finally carried out using standard Dacron grafts as controls.

Copper metallochaperones help prevent mis-metallation of proteins and ensure copper incorporation in specific Cu(I)-binding proteins by ligand-exchange reactions. The human copper metallochaperone (hCCS) for superoxide dismutase-1 (hSOD1) can interact via domain 1 with the carboxyl-terminal domain (CTD) of human β-secretase-1 (hBACE1). hBACE1 initiates the production of amyloid-β peptide (Aβ), the major constituent of senile plaques in Alzheimer’s disease (AD). One of the aims of this thesis was to identify the residues involved in mediating the interaction between hCCS and hBACE1 CTD and to investigate the role of copper in mediating this interaction using a yeast two-hybrid system. The data show that the Cys residues in the CXXC motifs of hCCS and hBACE1 CTD are essential for this interaction. The interaction is optimised by residues located close to the Cu(I)-binding sites. The hCCS/hBACE1 CTD interaction decreases in copper-deficient conditions. hBACE1 CTD was also shown to interact with the copper metallochaperone HAH1 involving the Cys residues in the CXXC motif of hBACE1 CTD. A model has been proposed based on the physiological implications of these data with respect to AD pathology. The interaction of hCCS with hSOD1 was also investigated using the yeast two-hybrid system. The mutation of the Cu(I)-binding residues in hCCS do not have a significant effect on the hSOD1/hCCS interaction. Interestingly, the hSOD1/hCCS interaction increases in copper-deficient conditions which can also have implications for AD pathology, as discussed in the proposed model. The yeast two-hybrid system was also used to study the interaction of the copper metallochaperone Atx1 from Synechocystis PCC 6803 (ScAtx1) with the metal-binding domains (MBDs) of the Cu(I)-transporting ATPases, CtaA (CtaAN) and PacS (PacSN). The data show that the residues on loop 5 of the ferredoxin-like fold of ScAtx1 (His61), CtaAN (Phe87) and PacSN (Tyr65) are important for mediating proteinprotein interactions. Swapping the loop 5 residue disrupted complex formation of ScAtx1 with CtaAN and PacSN. The mutation of ScAtx1 His61 to a charged residue or to a neutral Ala also weakens the interactions with the MBDs, demonstrating that His61 is the best choice of amino acid in this position to enable ScAtx1 to interact optimally with both of its target proteins. The possibility that ScAtx1 may form two structurally different complexes (side-to-side or head-to-head) with CtaAN and PacSN was also investigated. The data indicate that both CtaAN and PacSN can likely form a side-toside complex although the possibility that they can also form a head-to-head complex can not be excluded.

Alzheimer’s disease (AD) is characterised by cerebellar accumulation and aggregation of amyloid beta (Aβ), a cleavage product of the transmembrane amyloid precursor protein (APP). APP contains a range of functional domains in its large extracellular portion, among which are two copper-binding motifs and one zinc-binding motif. The copper-binding motifs are present in the amino-terminal region of APP and within the Aβ region of the protein and readily reduce Cu(II) to Cu(I), thus APP and its cleavage products are linked to copper metabolism and have been hypothesised to participate in cellular copper homeostasis. In this project human neuroblastoma cell lines SH-SY5Y were utilised to determine the effect of expressing a familial AD mutation on intracellular copper concentrations and possible functional alterations or deficits of enzymes that require copper as a co-factor. The familial AD mutation first found in a Swedish population was previously shown to increase the total amount of released Aβ. Direct phenotypic comparison between SH-SY5Y APPWT cell lines expressing endogenous levels of APP and APPswe Coupling native two dimensional liquid chromatography with metal analysis, SDS-PAGE and Principal Component Analysis identified one major copper and zinc containing pool as copper-zinc superoxide dismutase (SOD1) in soluble whole cell protein extracts. Comparative analysis of metal content between APP cell lines overexpressing APP carrying the Swedish mutation was performed in standard culture and manipulated copper concentrations. WT and APPswe cultures indicated a difference in metallation of SOD1 with copper. APPswe cultures displayed reduced metallation of SOD1, whereas SOD1 metallation with zinc remained unaltered. Functional analysis of copper-binding enzymes, such as SOD1 and cytochrome c oxidase (CCO), using standard biochemical approaches, identified lower activities for both enzymes during standard cell culture in APPswe cells. Upon treatment of cultures with increasing concentrations of exogenous copper APPWT enzyme activities remained unaltered but enzyme activities in APPswe cultures increased in direct correlation with increasing copper concentrations. Combined with phenotypic analysis of growth, survival and intracellular metal content it appears that APPswe cultures are copper deficient, but this can be overcome by copper supplementation. Lower copper accumulation also enables greater survival of APP swe Overall, these data suggest that APP cells in elevated copper. swe overexpression in SH-SY5Y cells results in functional copper deficiency which can be rescued by supplementation of cultures with exogenous copper. APPswe further confers resistance to copper toxicity not only via increased Aβ release, but also via increased copper delivery to enzymatic target proteins improving cellular antioxidant response and energy metabolism. These data are consistent with a function of APP in copper efflux either in a regulatory capacity or directly contributing to copper egress.

Cartilage destruction in arthritic disease is characterised by irreversible collagenolysis, resulting in loss of efficient joint function. Of the enzymes capable of hydrolysing native collagen fibrils, matrix metalloproteinase-13 (MMP-13) is the major collagenolytic MMP in osteoarthritis (OA), making this MMP an important diseasemodifying target. Addition of interleukin-1 (IL-1) and oncostatin M (OSM) to bovine nasal cartilage in explant culture results in a synergistic loss of the collagen matrix, accompanied by a dramatic increase in the expression of collagenase enzymes. Interleukin-4 (IL-4) is able to ameliorate this collagen degradation by the strong repression of IL-1+OSM-induced MMP-13 expression. The aim of this work was to determine the mechanism by which IL-4 abolishes IL-1+OSM-induced MMP-13 expression. Work examining the effect of IL-4 on the methylation status of CpG residues within the MMP-13 promoter failed to identify a role for epigenetic modification in the mechanism of action of IL-4. Subsequent cell signalling studies demonstrated Akt activation by IL- 4. Therefore, genome-wide microarray analyses of cytokine stimulated cartilage and chondrocytes was used to identify candidate Akt-interacting proteins involved in the repressive effects of IL-4 on MMP-13. Trb1 was identified as a novel gene potentially involved in the repression of MMP-13 by IL-4 via Akt. Gene silencing experiments in chondrocytes confirmed that transfection with Trb1 specific siRNA resulted in the rescue of IL-4 mediated repression of IL-1+OSM-induced MMP-13 expression, indicating an anti-inflammatory role for Trb1. Trb1 belongs to family of three tribbles proteins and additional studies to investigate the roles of other tribbles family members in MMP regulation identified Trb3 as having a potentially pro-inflammatory role in MMP regulation in chondrocytes. Silencing of Trb3 was reproducibly shown to abolish IL-1+OSM-induced MMP-13 expression. The novel data presented in this thesis indicate that tribbles proteins act as key regulators of catabolic and anabolic responses in chondrocytes. From these findings it could be hypothesised that alterations in functional levels of specific tribbles proteins may protect against aberrant MMP gene expression in chondrocytes. The identification of this potentially important regulatory mechanism of signalling pathways important in MMP-13 gene expression in chondrocytes could be translated into a tractable therapy for arthritis once the mechanism has been unravelled.